Neurotransmitter transporters can be classified into three different families based on their amino acid sequence similarities and the type of gradient used for transport. The first family, the Na+/Clxe2x88x92 dependent neurotransmitter transporter family, contains integral membrane glycoproteins that have twelve putative transmembrane domains and use sodium and chloride gradients to transport neurotransmitters across the plasma membrane. The members of this family can be subdivided into four subfamilies based on the type of neurotransmitter transported and specific molecular features of the proteins: (1) monoamine transporters, (2) GABA, betaine, taurine and creatine transporters, (3) glycine and L-proline transporters and (4) orphan clones. The second family, the Na+/K+ dependent glutamate transporter family, contains plasma membrane glycoproteins that have six to nine putative transmembrane domains, require K+ ions and use Na+ gradients as a driving force to transport the neurotransmitters across plasma membrane. The third family, the proton dependent vesicular transporter family, contains proteins powered by proton gradients to pump the neurotransmitter from the cytosol into synaptic vesicles.
The members of the monoamine subfamily of Na+/Clxe2x88x92 dependent neurotransmitter transporters include transporters for: dopamine (DA), norepinephrine (NE), serotonin (SER) and L-epinephrine(E). Most of the cloned monoamine transporters have been isolated from mammals. Monoamine transporters have also been isolated from fruit fly and frog. The cDNA sequences of these transporters made available by molecular cloning reveal features common to all monoamine transporters. The deduced amino acid sequences of all cloned monoamine transporters indicate the presence of twelve putative transmembrane domains, two highly conserved cysteine residues on the large second extracellular loop, conserved consensus sequences for two to four N-linked glycosylation sites and phosphorylation sites on the intracellular domains for protein kinase C (PKC), cAMP-dependent protein kinase (PKA) and Ca2+/calmodulin-dependent protein kinase (reviewed by Kitayama and Dohi, (1996) Jpn J. Pharmacol. 72:195).
Insect neurotransmission, like that in mammals, is mediated by several types of neurotransmitters: biogenic amines, amino acids, neuropeptides and nitric oxide. Among the monoamines known to participate in insect neurotransmission are: octopamine (OA), dopamine (DA), serotonin (SER), histamine and possibly tyramine (TA) (Osborne (1996) Pharmacol. Ther. 69:117).
OA, first discovered in the salivary glands of octopus, has been shown to be present in high concentrations in several insect nervous tissues. OA is a phenolamine, the monohydroxylic analogue of norepinephrine (NE). Based on OA""s similarity in structure to NE and the fact that OA appears to play many of the roles that NE plays in mammalian systems, it has been called xe2x80x9cthe insect norepinephrinexe2x80x9d. Several studies have shown that in insects, OA functions as neurohormone, neuromodulator and neurotransmitter (reviewed by Evans (1985) Octopamine. Comprehensive Insect Physiology. Biochemistry and Pharmacology. Volume 2, Kerkut et al., eds., Pergamon Place, Oxford).
In the insect nervous system, TA is generally considered the immediate precursor for OA. OA is synthesized from tyrosine by decarboxylation to tyramine and then subsequent xcex2-hydroxylation to OA (Evans (1985) supra). The quantification of OA and its precursors (tyrosine and TA) in the nervous systems of two lepidopteran insects, Trichoplusia ni and Manduca sexta, support the idea that TA represents the immediate precursor for OA. There are some indications that TA has a functional role distinct from OA. Various studies indicate a possible role for TA as neurotransmitter or neuromodulator, in spite of being the immediate precursor of the well established neurotransmitter, OA.
Among the monoamines present in the insect nervous system functioning as neurotransmitters, OA is the only one specifically active in insects and other invertebrates but not in vertebrates. This makes OA, OA receptors and OA transporters desirable targets for pest control strategies. Even though specific OA uptake systems have been functionally described in both insect tissue and synaptosomal preparations, the OA systems are heretofore relatively uncharacterized at the molecular level. There is a need in the field for characterization of components of the OA transport system that may serve as targets for insecticides. The present invention provides a nucleic acid encoding an insect OA transporter and related embodiments useful for the identification of insecticides.
The present invention is directed to an isolated nucleic acid encoding a lepidopteran octopamine (DA)/tyramine (TA) transporter. In a preferred embodiment the nucleic acid is isolatable from Trichoplusia ni. In another preferred embodiment the isolated nucleic acid has the nucleotide sequence of SEQ. ID. NO: 1. In another preferred embodiment the isolated nucleic acid has a sequence encoding the amino acid of SEQ. ID. NO: 2.
The present invention further provides vectors comprising a nucleic acid encoding a lepidopteran OA/TA transporter. Host cells comprising the vectors are also provided.
In another embodiment, the present invention provides a recombinant lepidopteran OA/TA transporter, and host cells, oocytes, kits and compositions comprising a recombinant lepidopteran OA/TA transporter. Methods of identifying agonists and antagonists to a lepidopteran OA/TA transporter are also provided.